Indigo is a chemical compound that can be used as a blue dye in the food and textile industry, for example, as a blue dye for jeans. Natural indigo was originally obtained from various unrelated plants, most notably Indigofera tinctoria, Isatis tinctoria, and Polygonum tinctorium, which were the principal sources of the blue dye since ancient times (perhaps as early as 2000 B.C.) until chemical methods were developed in the 19th century to make indigo.
Synthetic indigo has largely replaced natural indigo in the dye and pigment industry. Microorganisms having enzymes capable of producing indigo (from indole) have been reported.
The role of indole in tryptophan biosynthesis in plants is conventionally understood. In plants, indole is a transient intermediate of tryptophan biosynthesis where it is produced by the alpha subunit of a bifunctional tryptophan synthase enzyme (TSA) by cleaving indole-3-glycerol phosphate (I3GP) into indole and D-glyceraldehyde-3-phosphate (G3P). Indole is subsequently channeled to the active site of the tryptophan synthase beta subunit (TSB), where it is condensed with the amino acid serine to produce tryptophan and water. TSA-like genes associated with tryptophan biosynthesis have been reported in various plant species including maize, Arabidopsis, and Isatis tinctoria (also known as woad).
Indigo can be made from indole. Conversion of indole to indigo requires a hydroxylation of indole at position 3 that gives rise to indoxyl (i.e., 3-hydroxyindole), which spontaneously dimerizes in the presence of oxygen to form indigo. To date, neither a plant gene nor a plant enzyme that can convert indole to indoxyl has been identified.
In indigo-producing plants, indoxyl molecules can be prevented from spontaneously dimerizing into indigo by immediately converting indoxyl into indoxyl glycosides, such as indican (in the case of Indigofera tinctoria) and isatin B (in the case of Isatis tinctoria). To extract indigo (e.g., by vat fermentation), indoxyl glycosides can be hydrolyzed by beta-glucosidases (either from microorganisms or the plant) to release indoxyl, which then spontaneously forms indigo under aerobic conditions. Glucosyltransferases that convert indoxyl to indican have been purified and characterized in Polygonum tinctorium and Baphicacanthus cusia, while a beta-glucosidase gene that converts indican into indoxyl can be cloned from Polygonum tinctorium (Minami et al. 1999).
Different types of non-plant enzymes (e.g., from microorganisms or human liver) have been found to catalyze the oxidation of indole to indoxyl. But none have ever been identified in plants prior to the following disclosure. In microorganisms, these enzymes mainly oxidize other substrates, with indole being a fortuitous substrate. Such is the case for naphthalene (Ensley et al. 1983), toluene (Stephens et al. 1989) and tetralin (Moreno-Ruiz et al. 2003) dioxygenases, as well as for styrene (O'Connor et al. 1997), xylene (Mermod et al. 1986), and flavin-containing (Choi et al. 2003) monooxygenases, among other bacterial indole oxidases. In humans, certain P450 enzymes in the liver can oxidize indole (Gillam et al. 1999) besides other substrates, as a first step in detoxification of xenobiotics. Another human enzyme, indoleamine-2,3-dioxygenase, can also oxidize indole to form indigo, but only in the presence of hydrogen peroxide (Kuo & Mauk 2012), similar to the reaction catalyzed by chloroperoxidases in Streptomyces lividans that converts indole to indoxyl (Burd et al. 2001). Various enzymes have also been modified by mutation to enable indole oxidation into indigo and other related pigments, examples of which include toluene-4-monooxygenase (McClay et al. 2005), flavin-containing monooxygenase (Meyer et al. 2002), and at least two bacterial P450s (Li et al. 2000; Manna & Mazumdar 2010).
Indirubin (an anticancer compound useful for the treatment of chronic myeloid leukemia) can be produced by the dimerization of 3-hydroxyindole and isatin, an oxidation product of 3-hydroxyindole. No plant genes for producing free indole or indole hydroxylation have been identified.
Formation of indigo from indoxyl, either during vat fermentation of indigo-producing plants (Maugard et al. 2001) or during catalysis by microbial (Hart et al. 1992) and human enzymes (Gillam et al. 2000), can be often accompanied by formation of the red pigment indirubin. This pigment is an isomer of indigo, and formed by the coupling of indoxyl and isatin, a double oxidation product of indole. Indirubin is considered an impurity in indigo dye preparations, but is also the active constituent of an herbal remedy for leukemia containing Isatis tinctoria (Hoessel et al. 1999).